Synthetic molecules having enzyme-like characteristics of binding a substrate, catalyzing a reaction, and releasing the products to begin a new cycle of reaction (turning over) have been of increasing interest as man has tried to alter or improve upon nature.
One group of such molecules are the catalytic antibodies prepared and studied by Lerner and Schultz and their co-workers. For a review see, Lerner et al., Science, 252:659-667 (1991). These molecules utilize the antibody combining site to bind the substrate and catalyze the reaction. Antibodies are, however, very large molecules, having a molecular weight of about 160,000 D for the usually used IgG type, and even F(ab').sub.2 and Fab portions have molecular weights of about 105,000 and 52,000 D, respectively. In addition, antibodies are prepared from living cells, and to date, have not been prepared in bulk chemical amounts.
Polypeptides having a length up to about 50 residues are more readily prepared in bulk quantities. A few of such molecules have recently been reported to possess catalytic activity.
For example, Johnsson et al., Nature, 365:530-532 (1993) (See, also, commentary by DeGrado, Ibid., 488-489), reported results using two amphiphilic molecules they named oxaldie 1 and oxaldie 2 that catalyze the decarboxylation of oxaloacetate. These molecules had a length of 14 amino acid residues and were composed of lysine, alanine and leucine, with oxaldie 1 having a free N-terminal .alpha.-amino group and oxaldie 2 having its N-terminal .alpha.-amino group acylated.
Atassi et al., Proc. Natl. Acad. Sci., USA, 90:8282-8286 (1993) reported on two cyclic peptides named ChPepz and TrPepz that were said to mimic the catalytic triad (Ser/His/Asp) active site of .alpha.-chymotrypsin and trypsin, respectively. Both cyclic polypeptides contained 28 residues and differed in sequence by four residues. Polypeptide ChPepz was reported to hydrolyze an .alpha.-chymotrypsin substrate, whereas polypeptide TrPepz was said to hydrolyze a trypsin substrate. Subsequent workers were unable to reproduce the results reported by Atassi et al. See, Matthews et al., Proc. Natl. Acad. Sci., USA, 91:4103-4105 (1994); Corey et al., Ibid., 4106-4109; Wells et al., Ibid., 4110-4114.
An earlier report of Hahn et al., Science, 248:1544-1549 (1990) reported the synthesis and activity of a synthetic protein-like molecule named chymohelizyme-1 (CCHZ-1) composed of four parallel amphiphathic polypeptides covalently linked at their C-termini. Those four chains contained 17, 19, 21 and 15 residues. Molecule CHZ-1 also contained the .alpha.-chymotrypsin Ser/His/Asp catalytic triad, an oxyanion hole and a substrate binding pocket for acetyltryosine ethyl ester, an exemplary .alpha.-chymotrypsin substrate. This difficultly synthesized molecule was reported to exhibit affinity for .alpha.-chymotrypsin substrates and hydrolysis rates of about 0.01 that of the natural enzyme. More than 100 turnovers of the synthetic catalyst were reported.
Melittin is a 26-amino acid residue polypeptide that is the principal protein component of bee venom. Melittin has the amino acid sequence ##STR1## Melittin forms monolayers at air/water interfaces, dramatically lowering the surface tension of the water, and integrates into and disrupts natural and synthetic lipid membranes, leading to lysis of cells such as leukocytes, erythrocytes, lysosomes and mitochondria.
X-ray crystal structure data from melittin form I and form II crystals indicate that four melittin molecules associate with each other. Those data also indicate that residues 1-10 form a straight .alpha.-helix as do residues 13-26, with the axes of the two helices forming an obtuse angle with each other of about 120.degree.. Terwilliger et al., Biophys. J., 37:353-361 (1982); Terwilliger et al., J. Biol. Chem., 257:6010-6015 (1982). The two .alpha.-helices are joined by a so-called "hinge region" comprised of residues 11 and 12.
The self-assembled tetrameric molecules are arrayed in two pairs of two anti-parallel chains laid across each other. The uncharged C-termini of the crossed molecules are relatively near each other, whereas the charged N-termini of the crossed molecules are relatively further apart.
Melittin has also been shown to be unfolded at micromolar concentration, at low ionic strength, and at neutral pH values, whereas the tetrameric .alpha.-helical structure is adopted under conditions of relatively high ionic strength, at alkaline pH values (about pH 9) or at elevated polypeptide concentrations. These structural changes were determined using residue molar ellipticity values obtained by circular dichroism measurements at about 200-230 nm.
The present inventors and their co-workers have extensively studied melittin, and more particularly its deletion and substitution analogues. Those melittin analogues possess antimicrobial activity as well as hemolytic activity toward red blood cells (erythrocytes). See, Blondelle et al., Biochem., 30:4671-4678 (1991); Blondelle et al., Pept. Res., 4:12-18 (1991); Blondelle et al., in Peptides, Proceedings of the Twelfth American Peptide Symposium, (Smith et al., eds.) Escom, Leiden, pp. 433-434 (1992); and Blondelle et al. in Innovation and Perspectives in Solid Phase Syntheses, Epton ed., Intercept, Andover, pp. 121-127 (1992).
Blondelle et al., Blochim. Biophys. Acta, 1202:331-336 (1993) reported on the effects of replacing each residue except Trp-19 with a Trp residue on erythrocyte lysis (hemolysis). Those results indicated that Trp for Leu replacements at positions 9, 13 and 16 resulted in decreases in activity, whereas replacements of Gly-1, Lys-7, Thr-11, Gly-12, Pro-14, Ala-15, Lys-21 or Lys-23 provided significant increases in hemolytic activity. Those substitutions at positions Gly-1 and Lys-21 extended the .alpha.-helices at either terminus, whereas substitution of either Thr-11 or Gly-12 reduced the length of the linking or hinge region between the two helical chains, and replacement of Pro-14 replaced a helix-disrupting residue with a helix-extender. The increases in hemolytic activity were generally well correlated to increases in helical character as measured by residue molar ellipticities and retention times on reversed phase high performance liquid chromatograph (RP-HPLC) using a C.sub.18 -column.
In a more recent study, Perez-Paya et al., BioChem J., 299:587-591 (1994), a series of single residue substitution and omission melittin analogues were studied. These studies indicated that amphipathicity (amphiphilicity) as well as interchain distances and the orientation of hydrophobic residues were involved in the induction of stabilized tetrameric structures.
Over the last several years, developments in peptide synthesis technology have resulted in automated synthesis of peptides accomplished through the use of solid phase synthesis methods. The solid phase synthesis chemistry that made this technology possible was first described in Merrifield et al. J. Amer. Chem. Soc., 85:2149-2154 (1963). The "Merrifield method" has for the most part remained largely unchanged and is used in nearly all automated peptide synthesizers available today.
Although most peptides are synthesized with the Merrifield procedure using automated instruments, a recent advance in the solid phase method by R. A. Houghten allows for synthesis of multiple independent peptides simultaneously through manually performed means. The "Simultaneous Multiple Peptide Synthesis" ("SMPS") process is described in U.S. Pat. No. 4,631,211 (1986); Houghten, Proc. Natl. Acad. Sci., 82:5131-5135 (1985); Houghten et al., Int. J. Peptide Protein Res., 27:673-678 (1986); and Houghten et al., Biotechniques, 4(6):522-528 (1986), whose disclosures are incorporated by reference.
Illustratively, the SMPS process employs porous containers such as plastic mesh bags to hold the solid support synthesis resin. A Merrifield-type solid-phase procedure is carried out with the resin-containing bags grouped together appropriately at any given step for addition of the same, desired amino acid residue. The bags are then washed, separated and regrouped for addition of subsequent same or different amino acid residues until peptides of the intended length and sequence have been synthesized on the separate resins within each respective bag.
That method allows multiple, but separate, peptides to be synthesized at one time, since the peptide-linked resins are maintained in their separate bags throughout the process. The SMPS method has been used to synthesize as many as 200 separate peptides by a single technician in as little as two weeks, a rate vastly exceeding the output of most automated peptide synthesizers.
A robotic device for automated multiple peptide synthesis has been recently commercialized. The device performs the sequential steps of multiple, separate solid phase peptide synthesis through iterative mechanical-intensive means. This instrument can synthesize up to 96 separate peptides at one time, but is limited at present by the quantity of its peptide yield.
The interest in obtaining biologically active peptides for pharmaceutical, diagnostic and other uses would make desirable a procedure designed to find a mixture of peptides or a single peptide within a mixture with optimal activity for a target application. Screening mixtures of peptides enables the researcher to greatly simplify the search for useful therapeutic or diagnostic peptide compounds. Mixtures containing hundreds of thousands or more peptides are readily screened since many biochemical, biological and small animal assays are sensitive enough to detect activity of compounds that have been diluted down to the nanogram or even picogram per milliliter range, the concentration range at which naturally occurring biological signals such as peptides and proteins operate.
Almost all of the broad diversity of biologically relevant ligand-receptor (or affector-acceptor) interactions occur in the presence of a complex milieu of other substances (i.e., proteins make up approximately 5-10 percent of plasma, e.g. albumin 1-3 percent, antibodies 2-5 percent-salts, lipids/fats, etc. ). This is true for virtually all biologically active compounds because most are commonly present, and active, at nanomolar and lower concentrations. These compounds are also, in most instances, produced distant from their affection sites.
That a small peptide (or other molecule) can readily "find" an acceptor system, bind to it, and affect a necessary biological function prior to being cleared from the circulation or degraded suggests that a single specific peptide sequence can be present in a very wide diversity, and concentration, of other individual peptides and still be recognized by its particular acceptor system (antibody, cellular receptor, substrate, or the like). If one could devise a means to prepare and screen a large library of peptides containing up to milions of different sequences, the normal exquisite selectivity of biological affector/acceptor or other systems could be used to screen through vast numbers of synthetic oligopeptides.
Of interest in screening very large numbers of peptides is work by Geysen et al., which deals with methods for synthesizing peptides with specific sequences of amino acids and then using those peptides to identify reactions with various receptors. See U.S. Pat. Nos. 4,708,871, 4,833,092 and 5,194,392; P. C. T. Publications Nos. WO 84/03506 and WO 84/03564; Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 82:178-182 (1985); Geysen et al., in Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J. Immunol. Meth., 102:259-274 (1987); and Schools et al., J. Immunol., 140:611-616 (1988).
In U.S. Pat. No. 5,194,392, Geysen describes a method for determining so-called "mimotopes". A mimotope is defined as a catamer (a polymer of precisely defined sequence formed by the condensation of a precise number of small molecules), which in at least one of its conformations has a surface region with the equivalent molecule topology to the epitope of which it is a mimic. An epitope is defined as the surface of an antigenic molecule which is delineated by the area of interaction with an antibody molecule. The mimotopes are prepared on a series of plastic rods.
The above method, although elegant, suffers from several disadvantages as to peptides. First, owing to the small size of each rod used, a relatively small amount of each peptide is produced. Second, each assay is carried out using the rod-linked peptides, rather than the free peptides in solution. Third, even though specific amounts of each blocked amino acid are used to prepare the mixed amino acid residues at the desired positions, there is no way of ascertaining that an equimolar amount of each residue is truly present at those positions.
Rutter et al. U.S. Pat. No. 5,010,175 discloses the preparation of peptide mixtures that are said to contain equimolar amounts of each reacted amino acid at predetermined positions of the peptide chain. Those mixtures are also said to contain each peptide in retrievable and analyzable amounts and are constructed by reacting mixtures of activated amino acids in concentrations based on the relative coupling constants of those activated amino acids.
In addition, Furka et al., (1988, 14th International Congress of Biochemistry, Volume 5, Abstract FR:013) and (1988, Xth International Symposium on Medicinal Chemistry, Budapest, Abstract 288, p. 168) described the synthesis of nine tetrapeptides each of which contained a single residue at each of the amino-and carboxy-termini and mixtures of three residues at each position therebetween. These mixture positions were obtained by physically mixing resins reacted with single amino acids. The abstract further asserts that those authors' experiments indicated that a mixture containing up to 180 pentapeptides could be easily synthesized in a single run. No biological or other activity assays were reported. More recently, Furka et al., Int. J. Peptide Protein Res., 37:487-493 (1991) reported on the synthesis of mixtures of 27 tetrapeptides and 180 pentapeptides prepared by physically mixing reacted resin-linked peptides. Those peptides were synthesized with one or mixtures of three or four residues at each position along the chain. No biological or other activity results using those relatively simple mixtures were reported.
More recently still, Huebner et al. U.S. Pat. No. 5,182,366 described a similar process. Huebner et al. data provided for a mixture of tetramers having a glycine at position 2 from the amino- (N-) terminus and each of five different amino acid residues at positions 1, 3 and 4 from the N-terminus indicated that each of the residues at positions 1, 3 and 4 were present in substantially equimolar amounts and that glycine was present in its predicted amount. Similar data were also provided for twenty-five groups of pentamers, each of which had two known residues at the amino-termini and mixtures of five residues each at the remaining positions. No data were presented as to any activity or actually obtaining any selected peptide from the prepared mixtures.
A similar approach was also reported by Lam et al., Letters to Nature, 354:82-84 (1991). Those workers reported the preparation of millions of bead-linked peptides, each bead being said to contain a single peptide. The peptide-linked beads were reacted with a fluorescent- or enzyme-labeled acceptor. The beads bound by the acceptor were noted by the label and were physically removed. The sequence of the bound peptide was analyzed.
Recent reports (Devlin et al., Science, 249:404-405 [1990] and Scott et al., Science, 249:386-390 [1990]) have described the use of recombinant DNA and bacterial expression to create highly complex mixtures of peptides. More recently, Fodor et al., Science, 251:767-773 (1991), described the solid phase synthesis of thousands of peptides or nucleotides on glass microscope slides treated with aminopropyltriethoxysilane to provide amine functional groups. Predetermined amino acids were then coupled to predefined areas of the slides by the use of photomasks. The photolabile protecting group NVOC (nitroveratryloxycarbonyl) was used as the amino-terminal protecting group.
By using irradiation, a photolabile protecting group and masking, Fodor et al. reported preparation of an array of 1024 different peptides coupled to the slide in ten steps. Immunoreaction with a fluorescent-labeled monoclonal antibody was assayed with epifluorescence microscopy.
This elegant method is also limited by the small amount of peptide or oligonucleotide produced, by use of the synthesized peptide or nucleotide affixed to the slide, and also by the resolution of the photomasks. This method is also less useful where the epitope bound by the antibody is unknown because all of the possible sequences are not prepared.
The primary limitation of the above new approaches for the circumvention of individual screening of millions of individual peptides by the use of a combinatorial library is the inability of the peptides generated in those systems to interact in a "normal" manner with acceptor or substrate sites, analogous to natural interaction processes (i.e., free in solution at a concentration relevant to the receptors, antibody binding sites, enzyme binding pockets, reactant substrates or the like being studied without the exclusion of a large percentage of the possible combinatorial library), as well as the difficulties inherent in locating one or more active peptides. Secondarily, the expression vector systems do not readily permit the incorporation of the D-forms of the natural amino acids or the wide variety of unnatural amine acids which would be of interest in the study or development of such interactions.
Houghten et al., Letters to Nature, 354:84-86 (1991) reported use of physical mixtures in a somewhat different approach from those of Furka et al., Huebner et al. and Lam et al., supra, by using solutions of free, rather than support-coupled, peptide libraries or sets that overcomes several of the problems inherent in the above art. Here, 324 exemplary hexamer mixtures that contained more than 34 million peptides were first prepared whose N-terminal two positions were predetermined residues, whereas the C-terminal positions of the sets were equimolar amounts of eighteen of the twenty natural (gene-coded) L-amino acid residues. Binding studies were carried out using those 324 mixtures to determine which few provided optimal binding to a chosen receptor such as a monoclonal antibody or live bacterial cells. That study determined the two N-terminal optimal binding residues.
Another eighteen sets were then prepared keeping the optimal first two optimal binding residues, varying the third position among the eighteen L-amino acids used, and keeping the C-terminal three positions as equimolar mixtures. Binding studies were again carried out and an optimal third position residue was determined. This general procedure was repeated until the entire hexamer sequence was determined.
Similar studies are also reported in Pinilla et al. Vaccines 92, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pages 25-27 (1992); Appel et al., Immunomethods, 1:17-23 (1992); Houghten et al., BioTechniques, 13:412-421 (1992); Houghten et al., in Innovation and Perspectives in Solid Phase Syntheses: Peptides, Polypeptides and Oligonucleotides, R. Epton (ed.), Intercept, Ltd., Andover, pages 237-239 (1992); Houghten et al., in Peptides, J. A. Smith and J. E. Rivier (eds.), Proceedings of the Twelfth American Peptide Symposium, ESCOM, Leiden, pages 560-561 (1992); and WO 92/09300 published Jun. 11, 1992.
A still different approach was reported in Pinilla et al., BioTechniques, 13:901-905 (1992). In that report, a total of 108 free hexamer peptide mixture sets were prepared. Those sets contained one of eighteen amino acid residues at each of the six positions of the hexamer chains, with the other five positions being occupied by equimolar amounts of those same eighteen residues. Again, over 34 million different peptides were represented by those 108 sets (6 positions.times.18 residues/position).
Each of the sets was assayed for binding to a monoclonal antibody as receptor. The residue at each position that provided best binding results for that position provided a peptide sequence that was identical to the known epitope for that monoclonal. This process also provided sequences for other peptides that were bound almost as well by the monoclonal.
The peptide sets or libraries reported to date have themselves been ligands that bind to an acceptor (receptor), but once bound, carry out no reaction. It would thus be beneficial if the above peptide library approach could be expanded to encompass materials that possess an activity of their own so that an inherent property could be optimized, or a previously non-existent or minimal activity could be created or enhanced. The disclosure that follows relates to such a system that provides catalytic activity to a relatively short polypeptide.